Ischemic stroke results from interruption of brain arterial blood supply. The interruption can be permanent or transient. Since the requirements for oxygen and glucose in the brain are high and constant, interruption of blood flow induces extreme stress responses, leading ultimately to neuronal death. In addition, restoration of blood supply subsequent to ischemia induces a profound inflammatory response causing additional neuronal death (reperfusion injury). As ischemic stroke is one of the leading causes of death in both the United States and Europe, and survivors are faced with debilitating consequences, including permanent disability, therapies that might prevent or reduce ischemia-related neuronal death have been sought. Since much of the ischemia-related neuronal death and all of the reperfusion-related death occurs over the course of hours and days following the initial ischemic event, therapeutic intervention subsequent to a stroke has the potential to be highly beneficial. We and others have demonstrated that BH domain-containing survival factor, Mcl-1, is critical for neuronal survival, particularly under conditions of stress. We have also demonstrated that a ubiquitin ligase, SCFFbw7 targets Mcl-1 for ubiquitin-mediated proteolysis in neurons. Accordingly, RNAi- mediated silencing of the substrate-binding adaptor of this ligase, Fbw7, increases steady state levels of Mcl-1 in primary neuronal cultures and protects them from stress-associated apoptosis. We have therefore embarked on a program to identify small molecule inhibitors of SCFFbw7 in order to develop therapeutic interventions for pathological conditions resulting in neuronal apoptosis. Although cell death due to ischemic brain injury is not considered to be due solely to apoptosis, apoptotic neuronal death is thought to be a significant factor. To date we have identified a number of small molecule inhibitors that block stress-mediated apoptosis in primary cultured neurons with EC50s in the low- to mid-picomolar range. These stressors include hypoxia, OGD, and treatment with inflammatory cytokines. Preliminary pharmacokinetic analysis in the mouse of one compound (20aS20) indicates that it is stable in vivo and shows good CNS penetrance. Therefore, we plan to use this compound to determine if targeting SCFFbw7 has therapeutic efficacy in mouse models of ischemic brain injury. Demonstrating therapeutic efficacy would then justify a drug discovery program centered on inhibition of SCFFbw7.